The long-term goal of our research is to understand the molecular and structural basis of key initial events associated with human cytomegalovirus (HCMV) infection, including viral attachment, entry and assembly. HCMV is a leading viral cause of birth abnormalities and a life-threatening pathogen in immunosuppressed individuals. As the most structurally and genetically complex herpesvirus and one of the largest of all viruses, HCMV virion is composed of a glycoprotein-containing envelope, a tegument layer, and an icosahedral, bacteriophage-like capsid enclosing a double-stranded DNA genome. Our preliminary three-dimensional (3D) studies by electron cryomicroscopy (cryoEM) and electron cryotomography (cryoET) showed that HCMV tegument exhibits striking structural differences from other herpesviruses despite sharing a similar capsid and capsid assembly mechanism. Despite of its medical significances, little is known about the structures of HCMV tegument and glycoproteins owing largely to the lack of usable structural tools for such systems. We hypothesize that HCMV tegument and envelope proteins, as well as domains of the capsid proteins interacting with the tegument, have HCMV-specific structural and functional roles. The proposed research employs the newly emerging cryoET technology and harnesses the investigator's unique expertise in high-resolution cryoEM to tackle this important, vet under-investigated subject of HCMV infection. We will focus our research on visualizing key initial events of HCMV infection and molecular interactions essential to tegument assembly in 3D. Our aims are (1) to determine the structural and functional role of HCMV-specific tegument protein, pp150;(2) to identify the structural elements of major capsid protein that interact with SCP and pp150 by determining 5-7-A resolution structures of naked and tegumented HCMV capsids using cryoEM and structure-based mutagenesis;(3) to localize and determine the morphology of major envelope proteins, particularly glycoprotein B (gB) and gH and their interactions with receptors, by cryoET with antibody-labeling;and (4) to construct a 3D atlas of molecular interactions during HCMV attachment and entry by reconstructing 3D views of thin-sections of HCMV-infected cells. The results will be a series of much-needed 3D maps of HCMV entry and assembly at an unprecedented level of detail. Such new information will lead to better understanding of HCMV infection and ultimately benefit efforts of therapeutic intervention.